PROJECT SUMMARY AMPA-type glutamate receptors (AMPARs) are the major excitatory neurotransmitter receptor in the central nervous system. Dynamic regulation of the density, subunit composition and trafficking of AMPARs into and out of excitatory synapses is a key mechanism to facilitate changes in synaptic strength important for various forms of synaptic plasticity relevant to learning and memory. Over the last twenty years, work from Huganir Laboratory and others has made significant contributions to uncovering the molecular mechanisms underlying synaptic targeting of AMPARs that strengthens (long-term potentiation, LTP) or weakens (long-term depression, LTD) synaptic transmission. These studies have contributed to the prevailing model of LTP, whereby subunit-specific protein interactions and post-translational modifications within the cytosolic, carboxy (C)-terminus of AMPARs facilitate the trafficking of AMPARs to synapses and thereby enhance synaptic transmission. Recent work challenged this model and the requirement for the C-termini of AMPARs, as well as their interacting partners, to mediate LTP. It was found that not only could AMPAR mutants that lack the C- terminal domain rescue LTP in mice deficient all AMPARs (GluA1-A3), but so could GluK1, which belongs to the distinct, Kainate subclass of ionotropic glutamate receptors not normally found at these synapses. While these data remain a topic of debate, AMPA and Kainate receptors shared conserved structural architecture in their extracellular domains, raising the intriguing and less explored possibility that the extracellular domains of specific AMPAR subunits might also play key roles in regulating the synaptic targeting of the receptors. In this research proposal we will investigate the hypothesis that synaptic targeting of AMPARs required to induce long-term changes in synaptic strength is bi-directionally regulated by coordinated protein-protein interactions with the N- and C-termini. To test this hypothesis we have developed an assay that uses proximity- labeling with APEX2-tagged to the N- or C-termini of AMPAR subunits to perform an unbiased, proteomic screen to identify proteins that endogenously interact with either the extracellular or intracellular domains of GluA1- and GluA2-containing AMPARs during synaptic plasticity. Selected candidate proteins that demonstrate validated, dynamic and specific interactions with AMPAR subunits will be further characterized using overexpression and knockdown approaches to evaluate their role in regulating the synaptic targeting and function of AMPARs during synaptic plasticity. By identifying and characterizing new, functionally-relevant interactions between synaptic proteins and AMPARs, the proposed research will provide significant insight into the molecular mechanisms underlying plasticity relevant to learning, memory and higher brain function. Further, these studies have broad implications for brain disorders involving synaptic dysfunction as they might reveal novel therapeutic targets for the treatment of neurological and psychiatric diseases.